Cell systems having specific interaction of peptide binding pairs

Abstract

This invention relates to novel modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide-binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; said fusion protein comprising a first peptide of a peptide binding pair, or segment of said first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, said fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a luciferase gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

We claim:  
     
       1. A yeast cell comprising: 
       a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a known peptide binding pair that bind through extracellular interaction in their natural environment, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;  
       b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
       c) a luciferase gene activated under positive transcriptional control of the reconstituted transcriptional activation protein.  
     
     
       2. The yeast cell of  claim 1  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, and a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion. 
     
     
       3. The yeast cell of  claim 1  wherein the peptide binding pair comprises a ligand and a receptor to which the ligand binds. 
     
     
       4. The yeast cell of  claim 1  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor. 
     
     
       5. The yeast cell of  claim 1  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid. 
     
     
       6. The yeast cell of  claim 5  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors. 
     
     
       7. The yeast cell of  claim 6  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and transforming growth factor β (“TGF”). 
     
     
       8. The yeast cell of  claim 1  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA binding domain. 
     
     
       9. The yeast cell of  claim 8  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein. 
     
     
       10. The yeast cell of  claim 1  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  or  Pichia pastoris.    
     
     
       11. The yeast cell of  claim 10  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
       12. The yeast cell of  claim 1  wherein the luciferase gene is a Renilla luciferase gene. 
     
     
       13. The yeast cell of  claim 1  wherein the luciferase gene is a Photinus luciferase gene. 
     
     
       14. The yeast cell of  claim 1  wherein the first and second peptides of the peptide binding pair interact through extracellular interaction in their natural environment. 
     
     
       15. A yeast cell comprising: 
       a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a peptide binding pair, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;  
       b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the peptide binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;  
       wherein the nucleotide sequence encoding either the first or second heterologous fusion protein is present in an effective copy number of at least 5 copies per yeast cell and the nucleotide sequence encoding the other heterologous fusion protein is present at a copy number of 1 or 2 per yeast cell; and  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
       c) a luciferase gene activated under positive transcriptional control of the reconstituted transcriptional activation protein.  
     
     
       16. The yeast cell of  claim 15  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain and a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion. 
     
     
       17. The yeast cell of  claim 15  wherein the peptide binding pair comprises a ligand and a receptor for the ligand. 
     
     
       18. The yeast cell of  claim 15  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1 F, VP16, or a mammalian nuclear receptor. 
     
     
       19. The yeast cell of  claim 15  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid. 
     
     
       20. The yeast cell of  claim 19  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors. 
     
     
       21. The yeast cell of  claim 20  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF. 
     
     
       22. The yeast cell of  claim 15  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA binding domain. 
     
     
       23. The yeast cell of  claim 22  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein. 
     
     
       24. The yeast cell of  claim 15  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  or  Pichia pastoris.    
     
     
       25. The yeast cell of  claim 24  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
       26. The yeast cell of  claim 15  wherein the luciferase gene is a Renilla luciferase gene. 
     
     
       27. The yeast cell of  claim 15  wherein the luciferase gene is a Photinus luciferase gene. 
     
     
       28. The yeast cell of  claim 15  wherein the first and second peptides of the peptide binding pair interact through extracellular interaction in their natural environment. 
     
     
       29. A method of detecting the interaction of a first peptide and a second peptide of a peptide binding pair in the presence of a test sample, comprising: 
       (i) culturing at least one yeast cell, wherein the yeast cell comprises;  
       a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide of a known peptide binding pair that bind through extracellular interaction in their natural environment, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;  
       b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
       c) a luciferase gene activated under positive transcriptional control of the reconstituted transcriptional activation protein;  
       (ii) incubating the test sample with the yeast cell under conditions suitable to detect expression of the luciferase gene; and  
       (iii) detecting the interaction of the first peptide and the second peptide by determining the level of expression of the luciferase gene.  
     
     
       30. The method of  claim 29  wherein the yeast cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, and a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion. 
     
     
       31. The method of  claim 29  wherein the peptide binding pair comprises a ligand and a receptor to which the ligand binds. 
     
     
       32. The method of  claim 29  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor. 
     
     
       33. The method of  claim 29  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid. 
     
     
       34. The method of  claim 33  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors. 
     
     
       35. The method of  claim 34  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF. 
     
     
       36. The method of  claim 29  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain. 
     
     
       37. The method of  claim 36  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein. 
     
     
       38. The method of  claim 29 , wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  or  Pichia pastoris.    
     
     
       39. The method of  claim 38 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
       40. The method of  claim 29  wherein the luciferase gene is a Renilla luciferase gene. 
     
     
       41. The method of  claim 29 , wherein the luciferase gene is a Photinus luciferase gene. 
     
     
       42. The method of  claim 29 , wherein the first and second peptides of the peptide binding pair interact through extracellular interaction in their natural environment. 
     
     
       43. A method for determining whether a test sample interacts with a first or second peptide of a peptide binding pair, comprising: 
       (i) culturing at least one first yeast cell, wherein the first yeast cell comprises;  
       a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;  
       b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;  
       wherein the nucleotide sequence encoding the first heterologous fusion protein is present in an effective copy number of at least 5 copies per yeast cell and the nucleotide sequence encoding the second heterologous fusion protein is present at a copy number of 1 or 2 per yeast cell; and  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
       c) a luciferase gene activated under positive transcriptional control of the reconstituted transcriptional activation protein;  
       (ii) culturing at least one second yeast cell, wherein the second yeast cell comprises;  
       a) a nucleotide sequence encoding the first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;  
       b) a nucleotide sequence encoding the second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;  
       wherein the nucleotide sequence encoding the second heterologous fusion protein is present in an effective copy number of at least 5 copies per yeast cell and the nucleotide sequence encoding the first heterologous fusion protein is present at a copy number of 1 or 2 per yeast cell; and  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
       c) a luciferase gene activated under positive transcriptional control of the reconstituted transcriptional activation protein;  
       (iii) incubating a test sample with the first and second yeast cells under conditions suitable to detect luciferase activity;  
       (iv) detecting the luciferase activity produced by the first and second yeast cells; and  
       (v) comparing the detected luciferase activity of the first and second yeast cells, wherein lower luciferase activity in one of the yeast cells compared to the other yeast cell indicates that the test sample binds to the heterogeneous fusion protein encoded by the nucleotide sequence present at a copy number of 1 or 2 in that yeast cell exhibiting lower luciferase activity, thereby affecting the binding interaction of the peptide binding pair.  
     
     
       44. The method of  claim 43  wherein either or both of the first and second yeast cells further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain and a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, wherein at least one of the endogenous nucleotide sequences Is inactivated by mutation or deletion. 
     
     
       45. The method of  claim 43  wherein the peptide binding pair comprises a ligand and a receptor for the ligand. 
     
     
       46. The method of  claim 43  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor. 
     
     
       47. The method of  claim 43  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid. 
     
     
       48. The method of  claim 47  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors. 
     
     
       49. The method of  claim 48  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF. 
     
     
       50. The method of  claim 43  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain. 
     
     
       51. The method of  claim 50  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein. 
     
     
       52. The method of  claim 43  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  or  Pichia pastoris.    
     
     
       53. The method of  claim 52  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
       54. The method of  claim 43  wherein the luciferase gene is a Renilla luciferase gene. 
     
     
       55. The method of  claim 43  wherein the luciferase gene is a Photinus luciferase gene. 
     
     
       56. The method of  claim 43  wherein the first and second peptides of the peptide binding pair interact through extracellular interaction in their natural environment. 
     
     
       57. A method of simultaneously detecting the interaction of two different peptide binding pairs in the presence of at least one test sample, wherein the first peptide binding pair comprises a first peptide and a second peptide, and wherein the second peptide binding pair comprises a third peptide and a fourth peptide, comprising: 
       (i) culturing at least one yeast cell, wherein the yeast cell comprises;  
       a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a first transcriptional activation protein;  
       b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or segment thereof joined to a transcriptional activation domain of the first transcriptional activation protein;  
       c) a nucleotide sequence encoding a third heterologous fusion protein comprising the third peptide or segment thereof joined to a DNA binding domain of a second transcriptional activation protein;  
       d) a nucleotide sequence encoding a fourth heterologous fusion protein comprising the fourth peptide or a segment thereof joined to a transcriptional activation domain of the second transcriptional activation protein;  
       wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes the first transcriptional activation protein, and binding of the third peptide or segment thereof and the fourth peptide or segment thereof reconstitutes the second transcriptional activation protein;  
       e) a first luciferase gene activated under positive transcriptional control of the first reconstituted transcriptional activation protein;  
       f) a second luciferase gene activated under positive transcriptional control of the second reconstituted transcriptional activation protein; and  
       (ii) incubating the at least one test sample with the yeast cell under conditions suitable to detect luciferase activity; and  
       (iii) detecting the interaction of the first peptide and the second peptide by determining the level of expression of the first luciferase gene and detecting the interaction of the third peptide and the fourth peptide by determining the level of expression of the second luciferase gene.  
     
     
       58. The method of  claim 57  wherein the yeast cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, and a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion. 
     
     
       59. The method of  claim 57  wherein at least one of the peptide binding pairs comprises a ligand and a receptor to which the ligand binds. 
     
     
       60. The method of  claim 57  wherein the first or second transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor. 
     
     
       61. The method of  claim 57  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid. 
     
     
       62. The method of  claim 61  wherein at least one peptide of the peptide binding pairs is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors. 
     
     
       63. The method of  claim 62  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M. ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF. 
     
     
       64. The method of  claim 57  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain. 
     
     
       65. The method of  claim 64  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein. 
     
     
       66. The method of  claim 57 , wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  or  Pichia pastoris.    
     
     
       67. The method of  claim 66 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
       68. The method of  claim 57  wherein the first and second peptides of the peptide binding pair interact through extracellular interaction in their natural environment.